Method and device for culturing cells

ABSTRACT

In a method for culturing cells ( 2 ), cells ( 2 ) are placed, to form a cell layer, into a cell culture chamber which is formed in the interior of a support structure ( 1 ), the support structure ( 1 ) corresponding in its shape and size at least approximately to the shape to be formed by the cells ( 2 ), such as an implant or a prosthesis. Nutrients and/or oxygen are supplied to the support structure ( 1 ). The support structure ( 1 ) is furnished externally with a boundary layer ( 4 ) which is impermeable to cells.

The invention relates to a method for culturing cells, the cells beingintroduced into a cell culture chamber to form a cell layer. Theinvention also relates to an apparatus for culturing cells and a supportstructure therefor.

DE 199 35 643 A1 describes a method and an apparatus for culturingcells, cells being cultured on a support in a malleable cell culturechamber between films. The support is introduced in this case togetherwith the films into a container as bioreactor, nutrients and oxygenbeing supplied from outside.

A similar method and an apparatus therefor is also disclosed by DE 19719 751 A1. The cell growth in this case is more or less “uncontrolled”.The size of the apparatus is predetermined by the container in which thecell layer to be formed, or an implant, is located.

The object underlying the present invention is to provide a method andan apparatus for culturing cells, which method can be used in a veryversatile manner, in particular shapes formed from the cells, such asimplants or prostheses, being able to be produced in a very complexshape and in exactly definable size.

According to the invention this is achieved in a method for culturingcells, the cells, for formation of a cell layer, being introduced into acell culture chamber in such a manner that the cell culture chamber isformed in the interior of a support structure, the support structurecorresponding in its shape and size at least approximately to the shapeto be formed by the cells, such as an implant or a prosthesis, nutrientsand/or oxygen being supplied to the support structure, and the supportstructure being furnished externally with a boundary layer which isimpermeable to cells.

According to the invention the support structure is then the actualbioreactor itself, whereas previous bioreactors have only possesseddefined simple geometric shapes, for example round or square, flat orbottle-shaped. The external boundary layer which is impermeable to thecells gives an exactly defined cell culture chamber size and shape, thesize and shape themselves being predetermined by the support structure.This means that the support structure can be formed exactly in the shapeas the shape to be formed, for example the implant or the prosthesis, isto appear later in the final state. The implant to be manufactured ispreset in practice. Thus, for example, using a computer tomogram, bywhich, for example, a defect vertebra is recognized, this can bereproduced exactly as support structure. The cells are thencorrespondingly introduced into the support structure which has beenappropriately furnished with the boundary layer. Preferably, for this,the support structure is formed from a microporous, or else coarselyporous material. In this cases, the support structure can be constructedas a removable or else a convertible place holder material, so that thecell layer can form in accordance with the desired implant.

The boundary layer can be formed from a plastic which is impermeable tocells and, for this, can be applied, for example by injection or by adipping bath.

Materials which have proved to be suitable for this are, for example,liquid or viscous polymers, silicones, polyurethanes, proteins,alginates or resins.

Alternatively, the boundary layer can also be formed from a biologicalmaterial, for example a hydrogel or alginate. When an alginate is used,this can be polymerized in a calcium chloride solution and thus madeimpermeable to cells. For the subsequent removal and after completion ofthe implant, the polymerized alginate can be introduced into alow-calcium solution, whereby it redissolves.

If a self-dissolving boundary layer is not used, this can also beremoved mechanically after the completion of the cell culture method.

Advantageous developments and embodiments of the invention result fromthe remaining subclaims and from the exemplary embodiments describedhereinafter with reference to the drawing.

In the drawings:

FIG. 1 shows a vertebra as an implant to be formed, in highly simplifiedrepresentation;

FIG. 2 shows a dipping bath for the vertebra according to FIG. 1;

FIG. 3 shows the vertebra according to FIG. 1 having a boundary layer;

FIG. 4 shows a solution bath for the boundary layer;

FIG. 5 shows a container having a nutrient solution together with aplurality of implants;

FIG. 6 shows an implant in tubular bone shape;

FIG. 7 shows a partial implant for a heart valve;

FIG. 8 shows the implant shown in FIG. 3 in a nutrient circuit;

FIG. 9 shows the implant shown in FIG. 3 in a pressurizable container;

FIG. 10 shows a support structure in the form of a knee joint in acontainer; and

FIG. 11 shows the support structure according to FIG. 10 having anadditional meniscus structure.

FIG. 1 diagrammatically shows the shape of a vertebra, on the basis ofwhich the invention will be described in more detail below. Obviously,the vertebra is only to be considered as one possible example of aprosthesis or implant. The starting point for the vertebra is a supportstructure 1 which consists of a porous material, for example microporousor else coarsely porous. As material for the support structure 1, usecan be made of a stable, biodegradable or else remodelable material.Thus, for example, bone substitute material or else calcium phosphatecan be used. Plastics and hybrid structures, in which an industrialmaterial is combined with a biological material, are possible. What isessential is only that materials are used which are inert to cells 2 tobe introduced or which do not damage the cells which are introduced orpermit themselves to be modified by the cells 2.

For a defined cell culture chamber to be provided in the interior of thesupport structure 1 for the cells 2, and for the size and shape of theimplant to be formed to be maintained, it is necessary to ensure thatthe outer wall of the support structure 1 is impermeable to cells. Forthis, the support structure 1, before the introduction of the cells 2,can be immersed, for example, in a dipping bath 3 (see FIG. 2). Thedipping bath 3 can be, for example, a liquid or viscous polymer whichforms a boundary layer 4 (see FIG. 3) for the otherwise porous supportstructure 1. As polymers for forming a boundary layer, use can be madeof, for example, resins.

Instead of a polymer to encapsulate the support structure 1 by forming aboundary layer 4, alternatively use can be made of an alginate materialwhich is polymerized in a calcium chloride solution in the dipping bath3. Such a boundary layer 4 is biologically compatible.

The boundary layer 4 can be formed so as to be sealed absolutelytightly.

However, it is advantageous if it is formed to be at leastgas-permeable. In this case, oxygen can be introduced through theboundary layer 4. Likewise, it is also possible to form a boundary layer4 which is microporous in such a manner that nutrients also diffusethrough the boundary layer 4 into the interior of the support structure1, or a mass transfer with the support structure, that is to say thelater implant, takes place.

For the supply of cells 2, nutrient medium and if appropriate an oxygencarrier medium, for example fluoride solutions, blood or bloodsubstitutes, the support structure 1 can be furnished with a feedconnection 5. If, in the feed connection 5, nutrients are to beintroduced into the support structure 1, the support structure 1 canalso be furnished with an outlet connection 6, which provides throughflow. The feed connection 5 and the outlet connection 6 can beintroduced onto the support structure 1 upstream of the dipping bath 3or else downstream of the dipping bath 3, in which case, obviously, itis necessary to ensure that no impermeable boundary layer 4 is providedin the feed region and in the outlet region.

The support structure 1 can be constructed as a removable or elsemodifiable place holder material for the cells 2 to be introduced. Forinstance, a material can be used, for example for the support structure1, which material is dissolved on addition of cells inherent to thebody, for example by enzymes. In this manner, a matrix inherent to thebody is formed which is patient specific.

As boundary layer 4, use can also be made of a gel which forms a closedmembrane as a boundary layer.

After completion of the cell-formation process, or of the implant formedfrom the cells 2, the boundary layer 4 must be removed again. If it isof biological material, it can correspondingly be degraded againbiologically, as is the case, for example, with gels or alginates. Forthis, the support structure 1 having the boundary layer 4 can, inaccordance with FIG. 4, again be immersed in a bath 7 which degrades theboundary layer 4. When alginates are used, a solution can be used forthis which removes calcium so that the boundary layer 4 dissolvescorrespondingly. The boundary layer 4 can also be constructed in such amanner that it dissolves itself by an enzymatic or a hydrolytic process.The boundary layer 4 can also be vascularized or prevascularized (forexample by endoter- or stem cells).

When use is made of plastics or silicone which cannot be dissolved in asimple manner, the boundary layer 4 can if appropriate also be removedmechanically. To facilitate such removal, it is possible to dispose,between the support structure 1 and the boundary layer 4, anintermediate layer which does not bind to the material of the supportstructure 1. By means of this intermediate layer, the boundary layer 4may then be detached more readily. For this, use can be made of, forexample, a lipid layer, a protein layer and/or albumin layer, or othersoluble or detachable layers (biodegradable or erodable layers).

Instead of a supply of cells 2 via the feed connection 5, ifappropriate, the support structure 1 can alternatively be immersed, forexample, in an aqueous or pasty solution in which cells 2 together withnutrient solution are situated. In this case, the support structure 1 iscorrespondingly filled by absorption with cells 2 and nutrient solution.Then, the encapsulation by a boundary layer 4 in the dipping bath 3 isperformed.

Instead of a dipping bath 3, obviously a boundary layer 4 can also besprayed on or painted on as barrier for the cells 2.

FIG. 5 shows an embodiment in which a plurality of support structures 1furnished with boundary layers 4 are introduced into a nutrient mediumbath 9 so that the growth process starts. If appropriate, here also anoxygen feed into the nutrient medium bath 9 in addition can be provided.

FIG. 6 shows, as use example, a support structure 1 in the form of atubular bone which is likewise provided with the boundary layer 4internally and externally, and likewise can have a feed connection 5 andan outlet connection 6. Then, into the interior of the support structure1, it is possible to introduce stem cells which originate from the bonemarrow, which can be obtained, for example, by biopsy as the cells. Thestem cells then form, from the foreign support structure material,increasingly, in a time-dependent manner, their own bone material.Obviously, the material of the support structure 1 must then becorrespondingly soluble, for example formed from calcium phosphate.

FIG. 7 shows as a detail a heart valve having a stainless steel part,for example titanium part 10, around which is disposed externally thesupport structure 1, likewise a structure 1, likewise a feed connection5 and an outlet connection 6 being able to be provided. In this case,together with the titanium part 10, a support structure 1 is providedwhich reproduces the shape of a heart valve. Instead of the use oftitanium 10, a polyurethane prosthesis can also be used as a heartvalve.

FIG. 8 shows in principle the arrangement of a support structure 1together with cells 2 and a boundary layer 4 in a circuit 11 having apump 12 and a media reservoir 13. In the media reservoir 13, cellsand/or nutrient solution can be disposed. An oxygen carrier can also beincorporated into the circuit 11.

FIG. 9 shows the arrangement of a support structure 1 in a container 14into which opens out a feed line 15 for nutrients and/or oxygen, whichfeed line is connected to the feed connection 5. An outlet line 16 leadsout of the container 14 and is connected to the outlet connection 6.

In addition, the container 14 is furnished with a connection 17 forintroducing pressure medium and, if appropriate, also to outlet 18 forremoval thereof. As pressure medium, use can be made of a gas or aliquid medium.

This is because it has been found that the formation of a cell layer andthe cell growth are markedly improved if the cells 2 are exposed to apressure stress. In this manner, still better in-vivo conditions arecreated.

As a result of the exposure of the support structure 1 to pressure viathe pressurized container 14, exposure to pressure over a large surfacearea is achieved which simulates in-vivo conditions very well. In thiscase the pressure stresses can also be applied in alternation.

FIG. 10 shows for this a possible embodiment for joint cartilages whichare cultured on a support structure 1 which is to represent a kneejoint.

For the knee joint, which likewise can be a support structure 1′, forexample of tricalcium phosphate, cells 2′ can likewise be introduced ina manner not shown in more detail. If appropriate, for delimitationbetween the support structure 1′ and the support structure 1, in whichcartilage cells 2 are cultured, a boundary layer can be introduced, soas to introduce a clear separation between the cells 2 and 2′.

Via the feed connection 17, the interior of the container 14 is exposedto pressure by a pump 19. By means of the pump 19, changing pressure canbe introduced into the interior of the container 14. As shown, in thiscase an outlet connection 18 is not necessarily present.

As indicated dashed in FIG. 10, around the two support structures 1 and1′, in addition a protective film 20 can be disposed. The protectivefilm 20 can be provided for transport of the unit of the two supportstructures 1 and 1′ and can seal this unit correspondingly in a sterilemanner. The formation as elastically extensible film 20 ensures that thepressure stress applied by the pump 19 is transmitted to the supportstructures 1 and 1′.

Instead of tricalcium phosphate for the support structures 1 and 1′, inthe bone substitute field, collagens can also be used, in which case,for example, a meniscus can also be cultured. Likewise, connectivetissue structures, polymers such as polylactides or other chemicalstructures can be used. What is essential is only that from thesematerials, shapes can be constructed which correspond to the desiredimplant.

In addition, the container 14 may, in case of need, also be providedwith electrical connections by means of which, via connection lineswhich are not shown, electrical impulses can be applied to the cells 2and 2′, by which, likewise, still better in-vivo simulations can beachieved.

The embodiment shown in FIG. 11 essentially corresponds to the formdescribed in FIG. 10, for which reason the same reference symbols havealso been retained for the same parts.

The only difference is that, in addition, a meniscus structure 21 hasbeen applied over which, in turn, a support structure 1″ is situated.

For simplicity, in this case, a boundary layer 4 which is impermeable tocells 2 has been provided over the entire unit.

The boundary layer 4 can also be formed by the cells themselves whichare used to culture a membrane. On the support structure 1 then, forexample, connective tissue is formed as encapsulation. This can proceed,for example, by overgrowth processes with cells, for examplechondrocytes, fibroblasts or osteoblasts. These cells are then inpractice packaging cells and a boundary layer for the cells 2 to becultured.

1-30. (canceled)
 31. A method for culturing cells (2) comprising thesteps of; introducing the cells (2) into a cell culture chamber which isformed in an interior of a support structure (1), the cells (2) forminga cell layer, the support structure (1) at least approximatelycorresponding in shape and in size to one of an implant and a prosthesisto be formed by the cells (2); supplying at least one of nutrients andoxygen to the support structure (1); and externally furnishing thesupport structure (1) with a boundary layer (4) which is impermeable tocells:
 32. The method according to claim 31, further comprising the stepof forming the support structure (1) from a porous material which ispermeable to cells (2).
 33. The method according to claim 31, furthercomprising the step of forming the support structure (1) as a placeholder material which is one of removable or convertible by the cells(2).
 34. The method according to claim 33, further comprising the stepof forming the support structure (1) from phosphate.
 35. The methodaccording to claim 32, further comprising the step of furnishing thesupport structure (1) with the cells and a nutrient solution at a startof culturing, after which the boundary layer (4) is applied.
 36. Themethod according to claim 31, further comprising the step of forming theboundary layer (4) from one of a biological material and a syntheticmaterial.
 37. The method according to claim 36, further comprising thestep of forming the boundary layer (4) from a hydrogel.
 38. The methodaccording to claim 36, further comprising the step of forming theboundary layer (4) from an alginate which is polymerized in a calciumchloride solution and, after formation of the cell layer, removing theboundary layer (4) from the support structure (1) by a low-calciumsolution.
 39. The method according to claim 36, further comprising thestep of forming the boundary layer (4) by an overgrowth with cells whichform a membrane.
 40. The method according to claim 39, furthercomprising the step of forming the boundary layer (4) by one ofcartilage cells, fibroblasts, osteoblasts and chondrocytes.
 41. Themethod according to claim 31, further comprising the step of forming theboundary layer (4) so as to be gas permeable.
 42. The method accordingto claim 31, further comprising the step of applying the boundary layer(4) by one of spraying on a material which is impermeable to cells anddipping in a bath (3).
 43. The method according to claim 31, furthercomprising the step of introducing, between the support structure (1)and the boundary layer (4), an intermediate layer which does not bond tothe support structure (1).
 44. The method according to claim 43, furthercomprising the step of introducing, as the intermediate layer, one of alipid layer, glycoproteins, proteins, biodegradable or removable layers.45. The method according to claim 31, further comprising the step ofusing one of a liquid and a viscous polymer as the intermediate layer(4).
 46. The method according to claim 31, further comprising the stepof furnishing the support structure (1) with at least one inlet (5, 6)for at least one of oxygen and nutrients.
 47. The method according toclaim 31, further comprising the step of forming the boundary layer (4)so as to be mechanically removable.
 48. The method according to claim31, further comprising the step of forming the boundary layer (4) so asto be one or more of detachable, soluble and is vascularized orprevascularized.
 49. The method according to claim 31, furthercomprising the step of introducing a plurality of the support structures(1) into a nutrient solution.
 50. The method according to claim 31,further comprising the step of exposing the support structure (1) topressure by a liquid or gaseous medium.
 51. The method according toclaim 50, further comprising the step of inserting at least one supportstructure (1) into a container (14) which is exposed to a changing gasor liquid pressure by a pressure medium (19).
 52. The method accordingto claim 50, further comprising the step of placing a protective film(20) around the support structure (1) which forms a pressure chamberaround the support structure (1), and the protective film (20) beingexposed to pressure loads on a side facing away from the supportstructure (1).
 53. The method according to claim 31, further comprisingthe step of incorporating the support structure (1) into a nutrientcircuit (11) and is bound to an oxygen carrier.
 54. The method accordingto claim 53, further comprising the step of providing a nutrientreservoir (13) in the circuit (11).
 55. An apparatus for carrying out amethod for culturing cells (2) comprising the steps of; introducing thecells (2) into a cell culture chamber which is formed in an interior ofa support structure (1), the cells (2) forming a cell layer, the supportstructure (1) at least approximately corresponding in shape and size toan implant or a prosthesis, to be formed by the cells (2); supplying atleast one of nutrients and oxygen to the support structure (1); andexternally furnishing the support structure (1) with a boundary layer(4) which is impermeable to cells, the support structure (1) isfurnished with feeds and outlets (5, 6) and is used in a container (14)which is furnished with feeds and outlets (15, 16).
 56. The apparatusaccording to claim 55, wherein the support structure (1) is insertedinto a nutrient circuit (11).
 57. The apparatus according to claim 55,wherein the support structure (1) corresponds at least approximately inshape and in size to a vertebra.
 58. The apparatus according to claim55, wherein the support structure (1) corresponds at least approximatelyin shape and in size to a bone part.
 59. The apparatus according toclaim 55, wherein the container (14) is furnished with at least onepressure connection (17) for connection to a pressure source (19).
 60. Asupport structure for culturing cells (2) for formation of a cell layerin a cell culture chamber in an interior of the support structure (1),the support structure being formed from a porous material and furnishedexternally with a boundary layer (4) which is impermeable to cells.